Glycopeptide Analgesicsfor Combat Casualty Care. · Supplementary Notes (i.e., report contains...

Tuic I IM1\/CDCITV Chemistry and Biochemistry 1306 East University Blvd. IMt UlNlVLKbllY College of Science P.O. Box210041

OF ARIZONA«, Tucson, AZ 85721-0041 Tel: (520) 621-6354 Fax: (520) 621-8407

Office of Navel Research 875 North Randolph Street Arlington, VA 22203-1995

RE: Final Technical report for Award #N000T4-05-1-0807

To whom it may concern:

Attached please find the final technical report for award #N00014-05-l-0807, Non-Toxic Glycopeptide Analgesics for Combat Casualty Care. The period of performance was June 13, 2005 through June 30, 2008.

Please let me know if there is any further information you require to close this grant out.

Sincerely,

Robin Polt Professor of Chemistry & Biochemistry, BI05 The University of Arizona Tucson, AZ 85721 Office: (520) 621-6322 Cell: (520) 370-2654 FAX: (520) 621-8407 www.biochem.arizona.edu

cc: Naval Research Laboratory, Defense Technical Information Center and ONR REG San Diego-N66018

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1. Agency Use Only (Leave blank) 2. Report Date 5 December 2013

3. Report Type and Period Covered (i.e., annual 1 Jun 00 - 31 May 01) Final (13 July 2005 - 30 June 2008)

4. Title and Subtitle Glycopeptides as Analgesics: Non-Toxic Alternatives to Morphine for Combat Casualty Care

6. Author(s) Robin Polt, Ph.D.

5. Award Number ONR N00014-05-1-0870

7. Performing Organization Name (Include Name, City, State, Zip Code and Email for Principal Investigator) The University of Arizona Tucson, Arizona 85722-3308

E-Mail: poltSu. arizona . edu

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OFFICE OF NAVAL RESEARCH BALLSTON CENTRE TOWER ONE 800 NORTH QUINCY STREET ARLINGTON VA 22217-5660

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11. Supplementary Notes (i.e., report contains color photos, report contains appendix in non-print form, etc.) Attached 36 pg .pdf file (published review articles).

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13. Abstract (Maximum 200 Words) (abstract should contain no proprietary or confidential information) Background: Endogenous opiate peptides (enkephalins and endorphins) are more potent and specific than morphine and congeners. Specificity is determined by the "address segment" while binding is determined by the "message segment." Incorporation of carbohydrates into the address segment results in improved biodistribution and enhanced penetration of the blood-brain barrier (BBB). Thus, glycosylation of mu- or delta-selective peptides allows the resulting glycopeptides to be used as potent and safer alternatives to classical opiates such as morphine. Morphine and other muselective agonists are immunosuppressants, while the glycopeptide enkephalins are selective delta-agonists, which are known to be immunostimulants.

Hypothesis: Enkephalin transport and penetration of the blood-brain barrier can be determined by reversible binding to membranes, which may be manipulated by altering the amphipathicity of the address segment directly by introduction of hydrophilic sugar moieties, and lipophilic side chains.

Conclusion: Glycopeptide analogues of enkephalins are viable drug candidates. Obstacles regarding the synthesis and design of analgesics have been overcome. Further studies are required in order to meet FDA requirements. See attached .pdf file (14 pg and 22 pg reviews) for details.

14. Subject Terms (keywords previously assigned to proposal abstract or terms which apply to this award) Combat Casualty Care, Analgesic

15. Number of Pages 37 16. Price Code

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Unclassified

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Prescribed by ANSI Std. Z39-18 298-102

OTAN ORGANIZATION

Delta-Selective Glycopeptides Related to Enkephalin Produce Profound Analgesia with Reduced Side Effects in Mice

*'Robin Polt, 2Edward J. Bilsky and 3Richard D. Egleton 'The University of Arizona, Dept. of Chemistry, 1306 E. University Blvd., Tucson, AZ 85721

2The University of New England, Dept. of Pharmacology, Biddeford, ME 04005 3The University of Arizona, Dept. of Pharmacology, AZ Health Sciences Center, Tucson, AZ 85721

[email protected]

ANALGESICS BASED ON ENDOGENOUS NEUROPEPTIDES

The use of endogenous neuropeptides such as enkephalins and endorphins as drugs has remained an elusive goal since the 1970's. The principle reason for this is that peptides generally do not cross the blood-brain barrier, and are quickly degraded in the blood stream prior to delivery to opiate receptors in the brain. Animal research with glycosylated enkephalins and endorphins (dynorphins) indicates that potent analgesia is possible after intravenous or sub-cutaneous injection. Glycopeptides derived from delta-selective opioid agonists have 2-3X the potency of morphine, and lack many of the side effects associated with mu-agonists such as morphine. Morphine is still used on the battlefield for combat casualty care, and it is anticipated that further development of the glycopeptide analgesics will result in superior analgesics with greatly reduced side effects. Recent developments in this area are reported.

1.0 INTRODUCTION

There is no question that the American Civil War was a medical learning experience for the doctors involved in it. The American Civil War provided the setting for the first genuinely effective care of combat casualties with the introduction of field hospitals on or near the battlefield, and early treatment of casualties. (Figure 1) The pharmacopoeia of the day was not extensive by today's standards, but among the most effective agents were ether and chloroform anaesthetics used during amputations and other procedures, and morphine, used for the treatment of pre- and post-operative pain.1 In the South, the scarcity and expense of imported drugs forced the Confederate Army to establish several medical laboratories to manufacture drugs for military use.2

Empirical testing in military hospitals helped determine the clinical value of indigenous remedies. During this war morphine, both in its pure form and in various impure preparations of opium, gained its first widespread use on the battlefield, and in hospitals far removed from the field of battle. While there have been many advancements and refinements in combat casualty care in the intervening 130 years, morphine and its congeners are still used extensively, with many of the same unwanted side effects that were noted by the physicians of the 1860's. Chief among these unwanted side effects were respiratory depression and lowered blood pressure. It will never be known for certain, but it is very likely that opiates given to Stonewall Jackson in the course of his "diligent care" contributed to his death 8 days after the successful amputation of his left arm. It has recently been concluded that hemorrhagic shock and pneumonia, both possible sequellae of opiate administration, contributed to the death of this Confederate general, and consequently dealt a serious blow to the Confederate cause. The problems of opiate induced respiratory depression are followed closely by the problems associated with tolerance and physical addiction. So widespread was the problem of opiate addiction of former soldiers after the war that it was given the term "veteran's disease."

Paper presented at the RTO HFMSymposium on "Combat Casualty Care in Ground Based Tactical Situations: Trauma Technology and Emergency Medical Procedures ", held in St. Pete Beach,

USA, 16-18 August 2004, and published in RTO-MP-HFM-109.

RTO-MP-HFM-109 32-1

»ßUMiö

Delta-Selective Glycopeptides Related to Enkephalin Produce Profound Analgesia with Reduced Side Effects in Mice ORGANIZATION

Figure 1: Mathew Brady recorded the medical treatment of Union casualties in the American Civil War. Amputations were performed in the field (left), either with or without the benefit of chloroform or ether as an anaesthetic. If the wounded were lucky enough to make it to a hospital (right), pain was generally treated with various preparations of opium or morphine. At the time of the Civil War, and afterwards, opiate addiction was referred to as "the veteran's disease."

2.0 ENDOGENOUS OPIOID PEPTIDES

Long before the discovery of the opioid peptides, it was suspected that mammals produced an endogenous substance with morphine-like effects. Eventually, with the aid of immunocytochemistry, these substances were discovered, and eventually isolated and chemically characterized. Three major classes exist: the relatively large dynorphins and endorphins (sometimes collectively referred to as endorphins), and the much smaller enkephalins (methionine enkephalin and leucine enkephalin). All of these peptides are enzymatic hydrolysis products of much larger precursor proteins that have a wide variety of neuropeptides embedded within their sequences. The enzymatic cleavage of these precursor peptides into the neurotransmitters and neuromodulators that are secreted by neurons allows for many pathways for regulation, and is a complex issue that will not be discussed here.4

3.0 ENHANCED STABILITY AND BBB TRANSPORT OF GLYCOPEPTIDES

With the discovery of the endogenous opiate peptides in the 1970's, and the recognition of their high selectivity and potency, it was initially anticipated that a new pharmacopoeia based on met-enkephalin, leu- enkephalin, or ß-endorphin would emerge. Since these peptide opiates are degraded to pharmacologically inert amino acids, whereas morphine and similar alkaloidal pharmaceuticals produce a cascade of biologically active metabolites, it was logically (and correctly) assumed that peptide analgesics would possess a limited side effect profile. Problems associated with the physicochemical features of peptides, including their metabolic liability have been largely solved in the intervening years with the introduction of un-natural and/or D-amino acids, and by covalent modifications of the peptide backbone. Unfortunately, the pharmacodynamic behaviour of most peptides is still poor, and the blood-brain barrier (BBB) remains as a significant and largely unsolved deterrent to the effective delivery of peptide-based central analgesia. The BBB is not only a physical barrier represented by the tight junctions of the cells of the brain microcapillaries, but is also an enzymatic barrier caused by a broad spectrum of proteolytic enzymes and specific peptidases.

A significant advance was made in the transport of enkephalins was reported in 1994, when it was noted that glycosylated enkephalins penetrate the BBB to produce centrally mediated analgesia in mice after i.V. injection. A series of glycopeptides were synthesized5 with varying types of O-linked glycosides attached to

32-2 RTO-MP-HFM-109


Ser6 of a potent 8-selective sequence first studied by Roques6 (Figure 2). O-Linked glycosylation of the relatively lipophilic Leu-enkephalin C-terminal amide YdGFS*-CONH2 led to enhanced surfactant properties7

of the molecule, which in turn led to increased interaction with membranes and membrane mimics.8 Although these relatively short glycosylated neuropeptides had no defined conformation in aqueous solution (e.g. they existed as random coils), in the presence of sodium dodecyl sulphate (SDS) micelles or other membrane mimics they adopted a very restricted and well-defined set of conformations, as indicated by circular dichroism (CD) and 'H-NMR analysis.9

Tyr-dThr-Gly-Phe-Leu-Ser(Glycoside)-CONH2

H ° H ° f H °

"*"OH ° Y^

0

NH2

0|R

OH H07^/"0H

"OH .0

p-D-Glucose

OH H0K OH HO,

i \ | ß-Maltotriose \ I I OH ! c

ß-Maltose

OH HOJOH

oSH

*0H

ß-Lactose

OH

OH

ß-Melibiose °H0H OH

Figure 2: Glycosylated Enkephalin Analogues. Glycosyl hexapeptides were synthesized using solid-phase Fmoc chemistry. The Fmoc serine glycosides were incorporated as the peracetates, and synthesized using methods developed in the Polt group.

YdGFLS*-CONH2

Glycoside Glucoside Moiety

8 Binding

(nM) Binding

(nM)

MVD IC50

(nM)

GPI IC50 (nM)

1 (peptide control) — 2.1 7.5 2.7 25

2 (glucomorphin) ß-D-Glc 2.4 7.6 1.6 34

3 (maltomorphin) a-D-Glc-(l—>4)-ß-D-Glc 9.9 30.8 1.7 52.6

4 (maltotrimorphin) [a-D-Glc-( 1 —>4)]2-ß-D-Glc 3.8 15 7.7 71.7

5 (lactomorphin) ß-D-Gal-(l—>4)-ß-D-Glc 17.3 40 5.72 34.8

6 (biomorphin) a-D-Gal-(l—>6)-ß-D-Glc 5.6 36.6 6.06 43.8

Table 1: In Vitro Binding Activity and Functional Assays for Glycosylated DTLES. ICso's for 5- and (i-opioid binding were determined using displacement of 3H-labeled radioligands from rat brain homogenates. Functional assays were performed using electrically stimulated mouse vas deferens and guinea pig illium.

RTO-MP-HFM-109 32-3


■i

V. 3

2 600

f- 400

□ Tl/2 Brain ■ Tl/2 Plasma

(b) Compound

! u

ca as

(c)

2 3

Compound

Figure 3: Glycopeptide Stability and Transport. (a) Octanol-saline distribution for the unglycosylated peptide 1 and glycopeptides 2, 3, and 4. The addition of 1, 2, or 3 glucose units to the opioid peptide message significantly decreases lipophilicity. (b) The in vitro stabilities of the peptide and glycopeptides were measured in mouse brain and serum. Increased glycosylation led to significant increases in stability in both brain and serum. Brain stability increased with each additional glucose. However, in the serum, the stability of the trisaccharide was lower than that of the disaccharide. (c) Brain delivery of the peptides measured by in situ perfusion studies. Addition of glucose to the peptide significantly increased uptake. Uptake to the brain was improved further for the disaccharide, giving the maximal delivery. The trisaccharide produced no further increase in BBB penetration.

Classical pharmacological theories of BBB transport suggest that peptides are not lipophilic enough to diffuse into the brain.10 Glycosylation decreases lipophilicity even further. Despite this, greatly increased transport rates in rat brain have been observed for the glycosylated enkephalins (Figure 3). Previous studies with the glucoside 2 indicated that the increased transport was due to a saturable mechanism, thus further ruling out simple diffusion. Reversible interaction of the glycopeptides with the membrane is believed to promote transport through the brain capillaries by transcytosis." Several other possible modes of transport (simple diffusion and receptor-mediated processes) have been ruled out.12 Maximum transport rates (and maximum biological effects) are observed when the optimum degree of glycosylation is achieved. For this peptide, the disaccharide produces both the optimal transport and stability in vivo. In general, glycosylation leads to

32-4 RTO-MP-HFM-109


enhanced stability of the peptide "message" in both serum and brain. The identity of the individual sugars does, however, contribute to the overall biological effect, which is a product of both BBB transport rates and the stability of the peptide in serum, as well as metabolism and excretion by the liver and kidneys.

4.0 ANALGESIC EFFECTS OF GLYCOSYLATED ENKEPHALINS

I Early Phase (0-10 min)

Laie Phase (10-30 min)

3 u 3

c 1/3

(a) w/Compound 2 w/Compound 1 20 mg/kg, s.c. 20 mg/kg, s.c. (b)

S

(c) Carrageenan w/Compound 2

(2%, 20 til. i.paw) (30 nmol, i.paw)

13 6 0.3 1 3 Morphine Compound 2

(mg/kg, s.c.) (mg/kg, s.c.)

R=0.1VV, p=0.581

2—t-l-i—.4

-i-<6

Morphine

(d)

0.01 0.1 1

A i.c.v., nmoles (±95% CI)

Figure 4: Antinociception in vivo. The glycosylated enkephalins showed strong analgesic activity in tests of antinociception after peripheral administration, which are more clinically relevant than the tail flick assay, (a) Mouse formalin paw test, glycopeptide 2, s.c. (b) Mouse abdominal constriction test, glycopeptide 2, s.c. (c) Mouse paw inflammation test with carrageenan, glycopeptide 2, i. paw. Injection of glycopeptide 2 into the contralateral injection had no antinociceptive effects, (d) Antinociceptive effects (mouse tail flick) of glycosylated enkephalins (A50 values) after i.c.v. injection (X axis), and after i.v. injection (Y axis). Morphine has been included as a reference point, but has been excluded from the correlation values, shown on the upper left part of the diagram. The observed analgesia after I.V. injection correlates most strongly with glycopeptide stability (Fig. 3b), and brain transport values (Fig 3c), rather than the i.c.v. potency.

The extent of antinociception was shown to be comparable to, or even superior to the effects of morphine in mice after i.c.v. and i.v. administration'" using the warm water tail flick assay.14 The representative glycopeptides all produced full agonist effects in these assays with the potencies exceeding that of morphine

RTO-MP-HFM-109 32-5

Delta-Selective Glycopeptides Related to Enkephalin Produce Profound Analgesia with Reduced Side Effects in Mice OROANIZATI

on a (j.mol/kg basis in some cases. (Figure 4) Additional analgesic assays involving visceral, chemical and inflammatory pain states were also used to gauge the effectiveness of 2 and 5 after /.v. and s.c. injection.

3 mg/kg

d) s.c. Glucomorphin, 2

10 mg/kg 30 mg/kg

I fc:-:-.r:v .:-■ ■^••i| IKS' :v:; ?'^'

10 mg/kg 30 mg/kg 60 mg/kg

Figure 5: Non-analgesic effects of opioids on mice. Both mice have received equi-analgesic (A90) doses of drug, (a) and (b) Glycopeptide-based analgesia did not induce Straub tail, (c) Morphine- induced analgesia induced large increases in locomotor activity, Stereotypie circling, compared to equi-analgesic doses of glycopeptide 2 (d).

32-6 RTO-MP-HFM-109


Two well-known effects of morphine in rodents are increases in locomotor activity' l6 with Stereotypie patterns of movement,17 and increases in muscular rigidity, including Straub tail.18 Unlike morphine and other u-selective opioids, at equivalent s.c. A90 antinociceptive doses, or even supramaximal doses, the glycopeptide analgesics produced minimal increases in locomotor activity, and did not produce Straub tail (Figure 5). These results were confirmed in two different strains of out-bred mice.

5.0 MECHANISM OF TRANSPORT

Evidence obtained from in vivo19'10 as well as in vitro experiments9 with the glycopeptides are consistent with an endocytotic mechanism of transport. Receptor mediated transport and diffusive mechanisms have been ruled out, and further work strongly suggests that adsorption to the endothelial membrane of the brain capillaries is required for BBB transport. While the drug must adsorb strongly to the membrane in order to undergo endocytosis or transcytosis, this must also be a reversible adsorption, otherwise the drug will bind tightly to the first membrane it sees, resulting in poorer transport. This concept is demonstrated clearly with the amphipathic oc-helices, 14, 15, and 16. (Table 3)

Our work began with glycosylated enkephalins that were designed to have potent 5-agonist activity, but still have appreciable ^.-agonist activity. While it is possible to produce some analgesic effects through the 8- receptor alone, previous work has shown that |>agonists are much more effective in this regard. It was hoped that mixed 8/u-agonists would show reduced side effects, relative to u.-selective agonists, e.g. morphine. Other researchers have proposed u-agonist/5-antagonists as drug candidates for analgesia with reduced side- effects.21 An important aspect that is not fully understood is the role that "address" segments play in determining receptor selectivity.

Helices are the most commonly occurring secondary structural elements in globular proteins, accounting for one-third of all the residues.22 Linus Pauling first proposed the a-helix as an important motif of secondary structure in proteins in 1948,23 interestingly, without any experimental evidence.24,25 Segrest first theorized the amphipathic (a.k.a. amphiphilic) helix to be an important structural motif of integral membrane proteins in 1974.26 It is estimated that over 50% of all a-helices in nature are amphipathic.27 These proteins are unique in that they possess hydrophobic and hydrophilic parts either by primary structure (highly hydrophilic N- terminus and hydrophobic C-terminus) or by secondary structure, with polar residues pointing one to face and the non-polar residues on the opposite face. This allows them to "float" in a cell membrane, exposing the hydrophilic side to the aqueous exterior of the cell and the hydrophobic side to the lipophilic membrane.28,29

This peptide-membrane interaction is believed to be important for two reasons. First, the amphipathic nature of the helix can help guide a drug or hormone to its specific receptor by narrowing the receptor search from a 3-dimensional search to one in 2-dimensions. Surface-assisted "reduction-of-dimensionality" calculations, performed by Polya in 1921, were examined by Max Delbrück in which he quantitatively demonstrated the viability of this theory.30 Assuming that no other forces are at work {e.g. convection), and that the membrane is fluid, the probability of a substrate finding its corresponding receptor is much better in 2-dimensions {e.g. a cell surface) than in 3 {e.g. in solution)— almost 100% when the search is reduced to 2-dimensions.

Second, membrane insertion may allow the portion that interacts with the receptor (pharmacophore or "message") to be fixed in a specific geometry. By restricting mobility in the membrane near the binding site, the amphipathic a-helix can dramatically alter the peptide-receptor interaction/1 In addition, membrane insertion can also induce a specific conformation in the ligand, different from its solution conformation. It seems clear that the bioactive conformation of a peptide is the membrane-bound conformation, and that membrane insertion is actually the first step in receptor activation.

RTO-MP-HFM-109 32-7

Delta-Selective Glycopeptides Related to Enkephalin Produce Profound Analgesia with Reduced Side Effects in Mice OROANIZATION

The endogenous neuropeptide ß-endorphin is a 31-residue naturally occurring opioid peptide. The first 5 residues of ß-endorphin are identical to Met-Enkephalin. It has been shown that the a-helical structure of C- terminal region of ß-endorphin plays a role in the receptor binding & opiate activities, and resistance to proteolysis.32 Kaiser33 proposed that ß-endorphin consists of the [Met5]-enkephalin peptide sequence at the N-terminus, a hydrophilic linker region from residues 6—12, and an amphiphilic helical region between the residues Pro13 and Gly30, which were assumed to be "helix breakers." This hypothesis has been supported by the conformational analysis of a number of ß-endorphin mimics with artificial C-terminal helical regions with amphipathic character 34 35,36 All of the analogues were a-helical by CD measurements, as the monomer or oligomers, and showed strong opioid agonism in vitro when compared to natural ß-endorphin. These studies clearly suggest that amphipathicity of the entire peptide is more important than the identity of specific amino acids present in the helical C-terminus.37 This has been further supported by the work of Kyle,38 who synthesized several potent peptide analogues containing the cc-helix-promoting residues a-aminoisobutyric acid (Aib) and N-methyl alanine (MeAla) near the C-terminal region of nociceptin, the natural ligand for the recently identified opioid receptor-like 1 receptor (ORL-1). According to Schwyzer, the N-terminal "message" is steered toward certain receptors and away from others by the C-terminal "address" segment, which interacts with the membrane to orient the message with respect to the receptor.

Dynorphin A (1-17) is an endogenous opioid heptadecapeptide which binds preferentially to the K opioid receptor.40 Dynorphin consists of a N-terminal message identical to Leu-enkephalin, and an address sequence that imparts selectivity for K receptors.41 Dynorphin A is believed to adopt an extended and/or random coil structure as determined by various spectroscopic measurements.42'43,44'45'46 In the presence of DPC micelles Dynorphin A is believed to contain a less ordered N-terminus, a well defined a-helix segment spanning between Phe4 and Pro10 or Lys"and a ß-turn from Trp14 to Gin17.47 Based on NMR results, the authors concluded that both the a-helix and the C-terminal ß-turn may be a consequence of dynorphin's interaction with the micelle, and may be important structural features of the full-length peptide when bound to the cell membrane in vivo. The a-helix could have multiple roles in positioning the amphipathic helix for interaction with the receptor, as amphipathic helices have many roles at interface.

Helix Glucoside

Glycopeptide Sequence Retention

Time (RP-HPLC)

% Helicity

(CD)

i.c.v. Analgesia IC50 (p/coMol)

7 YtGFLGELAS*KWFNALE 8.85 min 69% insoluble

8 YtGFLGELAS*KWFNALES* 7.95 " 55% 270

9 YtGFLGELAS *K WFN ALES * F 9.91 " 53% insoluble

10 YtGFLGELAS*KWFNALES*FW 12.48" 68% insoluble

11 YtGFLGLLKS*FAES*WS*NF 6.69 " 34% -30

12 YtGFLGKS*FAELWS*NFLS* 5.35 " 14% -30

13 YtGFLGLLKS*FWES*WS*NF 8.25 " 37% -30

Table 2: Glycosylated Endorphin Analogues.

32-8 RTO-MP-HFM-109

m NATO y OTAN


Helix Glucoside

Glycopeptide Sequence (3rd Generation)

Retention Time

(RP-HPLC)

Per Cent Helicity

(CD)

MVD IC50

(nM)

GPI IC50 (nM)

14 YtGFL(P)NLBEKALKS*L-CONH2 31.57 21 34.5 63.1

15 YtGFL(ßA)NLBEKALKS*L-CONH2 33.50 26 23.0 354

16 YtGFL(GG)NLBEKALKS*L-CONH2 30.30 14 18.8 196

— Morphine — — 258 54.7

a)

Table 3: Glycosylated Endorphin Analogues.

4U

N

0.5

0.45

( m.'' '5^^3rd Generation Helices 0.4

^^^ 0.35

0.3

0.25

2nd Generation Helices 0.2

rT . ^ 0.15

' ^-~_ -^ 1st Generation Helices 0.05

) 10 20 30 40 50 60 70 8 0 0

%- Helicity b)

Sucose

Figure 6: (a) Plot of Retention Time vs Degree of Helicity. (b) Mouse BBB Transport Data.

The first- and second-generation endorphins were also based on the 8-selective YdGFL- opioid message. Formed by simple truncation, the first generation helices, 7—10, were designed to probe the minimum length for helix formation. Essentially, we overshot the target, and all of these compounds were extremely helical, but they were not water soluble enough to work with, with the exception of helix 8. This compound possessed appreciable antinociceptive activity, however.4 All of these compounds were quite soluble in the presence of SDS micelles. Since these compounds are so stable in their helical form, they probably form aggregates, and fall out of solution in the absence of the detergent. The second generation helices, 11—13, were designed to be less lipophilic, and consequently were more water soluble, and showed much less helicity in the presence of micelles.49

The third-generation helical endorphin-based glycopeptides, 14—16, used the same 5-selective peptide DTLET first studied by Roques, and showed much superior properties, both in the chemistry lab and in the mouse. Using in situ methods in the mouse, not rat studies as before, Egleton was able to measure BBB transport rates independently of analgesia, and Bilsky has been able to demonstrate the analgesic effects of these larger glycopeptides using i.e.v. tail flick results in the mouse.30 Initial studies with these glyco- hexadecapeptides indicated that BBB transport rates were determined by the amphipathic nature of the glycopeptides,51 rather than the lipophilicity of the compound, per se,52 and that they actually show BBB transport rates that are similar to, or better than the shorter enkephalin analogues.

RTO-MP-HFM-109 32-9


These endorphin analogues have the same N-terminal YdGFL- opioid message contained in the enkephalin analogues 1—6, and the same C-terminal amide address sequence ~NLBEKALKS*L-CONH2, where B is the helix-stablizing a-aminoisobutyric acid (Aib) residue, and S* is the serine glucoside residue. The "linker region," which is intended to "break" the helix, and prevent propagation of the helical address into the opioid message, is different in the three glycopeptides: 14 => proline, 15 => ß-alanine, and 16 => glycylglycine.

Figure 7: Lipid Bound Helix. One structure of glycopeptide 14 in the presence of micelles, as determined by NOE-constrained molecular dynamics calculations. The message segment is labelled in yellow, and the helix indicated with the overlaid ribbon. The structure on the right has the hydrophobic (blue) and hydrophilic (red) surfaces labelled. The structures were rendered with the MOE software package.

Figure 8: Lipid Bound Helix-Bend. One structure of glycopeptide 17 in the presence of micelles, as determined by NOE-constrained molecular dynamics calculations. The message segment is labelled in yellow, and the helix indicated with the overlaid ribbon. The structure on the right has the hydrophobic (blue) and hydrophilic (red) surfaces labelled. The structures were rendered with the MOE" software package.

While the data presented in Figure 6 is interesting, and perhaps even compelling, it is also clear that one cannot only use the degree of helicity to predict amphipathicity. NMR evidence, in conjunction with Monte Carlo calculations (NOE constraints not discussed here) shows that the glycopeptides bind to micelles, and

32-10 RTO-MP-HFM-109


adopt a very restricted set of conformations. For the helices 14, 15, 16, and the disaccharide 17 (not pictured in Table 3, but is the top-most data point in Figure 6a) we see two membrane bound conformational ensembles, one that is very helical, (e.g. Figure 7) and one that has a helix-bend motif (e.g. Figure 8), but is none-the-less very amphipathic. The peptide sequence for 17 is the same as the sequence for 14, but the compounds differ in that 14 is glycosylated with the ß-D-glucoside, and 17 is glycosylated with the disaccharide ß-lactose. These two compound both show the same conformations in their micelle-bound ensembles based on NMR, and similar helicities based on CD, but slightly different population densities.

While it there is still much to be learned about the details of both the transport and binding processes of the amphipathic glycopeptides, an important principle has emerged concerning transport. It seems clear that one must have a glycopeptide that essentially has two states: 1) A state defined by one or more membrane-bound conformations that permit or promote endocytosis. 2) A state defined by a water-soluble, or random coil state that permits "membrane hopping." The key to efficient transport is to balance these two states so that the compound is neither retained in the membrane, or held in solution so that it cannot undergo adsorptive endocytosis. It may also be true that aggregation of glycopeptides on a membrane surface may actually initiate and promote endocytosis.

6.0 CONCLUSIONS

Based on the results obtained so far, it would seem that further pre-clinical studies are warranted to test the viability of the glycosyl enkephalin analogues (e.g. compounds 2, 5 or 6) as a replacement for morphine on the battlefield. Anecdotal studies in mice suggest that these compounds possess an extremely low level of toxicity, even at super-analgesic doses. The notion that one could administer a large sub-cutaneous dose of a non-toxic glycopeptide that would have prolonged analgesic effects without respiratory depression or the risk of overdose is particularly appealing. Further research needs to be completed in order to quantify the effects of the glycosylated 5-agonists on respiration and blood pressure, particularly in hypovolemic animals to gauge the propensity of these compounds to induce hemorrhagic shock. Complete absorption, metabolism and excretion studies (ADME) need to be completed, and oral bioavailability needs to be explored. The fact that the glycosylation strategy seems to be effective with the much larger endorphin analogues (e.g. compounds 14 and 15) suggest that this approach may have general applicability to BBB transport of non-analgesic (or even non-opioid) neuropeptides, which could lead to novel treatments for anxiety, stress-related disorders and depression.

ACKNOWLEDGEMENTS

We would like to thank Professors Victor J. Hruby, Frank Porreca, Henry I. Yamamura for helpful advice throughout the years. We also thank Dr. Muthu Dhanasekaran, Dr. Michael M. Palian, Dr. Scott A. Mitchell, Dr. Lajos Z. Szabo, Dr. Neil Jacobsen, Mr. Charles M. Keyari and Ms. Larisa Yeomans for experimental results, and the Office of Naval Research (N00014-02-1-0471) for financial support.

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[29] Amphipathic helix motif: classes and properties. Segrest, J.P.; De Loof, H.; Dohlman, J.G.; Brouillette, CG.; Anantharamaiah, G. M. Proteins: Struct., Fund, Genet., 1990, 5(2), 103—17.

[30] Adam, G.; Delbruck, M. in: Structural Chemistry and Molecular Biology. Rich, R.; Davidson, N., Eds., Freeman & Co., San Francisco, 1968, p. 198—-ff.

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[32] A Protein-Lipid Model Of The Opiate Receptor, Lee, N.M.; Smith, A.P. Life Sei. 1980, 26, 1459—64.

[33] Design and synthesis of a model peptide with ß-endorphin-like properties. Taylor, J.W.; Osterman, D.G.; Miller, R. J.; Kaiser, E.T. J. Am. Chem. Soc, 1981,103, 6965—6966.

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[44] Solution conformation of the pituitary opioid peptide Dynorphin (1-13). Maroun, R.; Mattice, W.L. Biochem. Biophys. Res. Commun. 1981,103, 442—446.

[45] Solution structure of Dynorphin A (1-17): a NMR study in a cryoprotective solvent mixture at 278 K. Spadaccini, R.; Crescenzi, O.; Picone, D.; Tancredi, T.; Temussi, A. J. Peptide Sei. 1999, 5, 306—312.

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[50] Systemic delivery of neuropeptides to the brain by glycosylation. Egleton, R.D.; Bilsky, E.J; Dhanasekaran, M.; Lowery, J.; Davis, P,; Porreca, F.; Yamamural, H.I.; Yeomans, L.; Keyari, CM.; Polt, R. unpublished.

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REVIEW | SPECIAL FOCUS: DRUGS OF ABUSE AND

TREATMENT OF ADDICTION

Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins

Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood-brain barrier. Extremely potent and selective opiate agonists have been developed from endogenous peptides, some of which show great promise as drug candidates.

The success rate of CNS drug development is lower than that of other therapeutic areas [1], There are multiple reasons for this dearth of new drugs, including the sheer complexity of the brain and its neuropathologies; a propensity for CNS drugs to cause CNS-mediated side effects that limit dosing and compliance; a lack of validated biomarkers to inform whether a given neurotherapeutic agent is engaging the target in sufficient concentrations to modulate the CNS target; and the presence of the blood-brain barrier (BBB), across which CNS agents need to penetrate. Among these challenges, the BBB is considered to be most problematic for peptide or protein-based therapies p-4]. However, peptides and proteins do offer distinct advantages for developing efficacious and well-tolerated treatments for CNS diseases, such as chronic pain, Alzheimer's disease and Parkinson's disease. These advantages include intrinsic affinity and selectivity of the peptide for native receptors and the metabolism of the peptide into smaller fragments and amino acids (versus the variety of active metabolites seen with small molecules). In addition, peptides and proteins can bind to multiple sites across a receptor protein, offer- ing greater opportunity to fine tune the receptor-effector response [5,6]. Historical challenges for peptide and protein drug development are being addressed, including modifications that increase stability, techniques that increase yields and lower total synthetic costs, and technology to improve tissue targeting, including access to the CNS. A number of monographs have been written since the beginning of this century that describe formulations and articulate solutions of increasing sophistication to address the problems of peptide-based drugs [7-9].

Opioid receptors The classical opioid receptors [10] are divided into three subtypes, the u receptor (MOR or MOP), the 8 receptor (DOR or DOP) and the K receptor (KORor KOP). Another receptor, the nociceptin or orphanin receptor (NOP or ORL1), is widely distributed in the CNS, and is clearly related to the opioid receptors in terms of its molecular biology but is generally not regarded as an opioid receptor as it does not respond to classical opioid agonists or antagonists. IUPHAR recommends use of the terminology MOP, DOP, KOP and NOP, replacing the older recommendation for OP, and OP2, for example [201]. All of the opioid receptors have been cloned from various species, including mouse, rat and human. Opioid receptors are GPCRs that consist of highly homolo- gous seven-transmembrane helical domains, and are linked with extracellular peptide loops of very limited size (FIGURE I) [11,12].

The MOP receptor [13] is distributed pre- synaptically in various brain regions, including the limbic structures, the brainstem (i.e., the periaqueductal grey area) and in the superficial dorsal horn of the spinal cord. It was initially characterized in functional smooth muscle preparations of the guinea pig ileum (GPI). This receptor remains the principal target of opioid analgesics currently in clinical use, with the u referring to morphine. The MOP receptor is widely distributed in other areas of the brain, as well as non-CNS tissues, most relevantly in the immune system, and the cardiovascular and gastrointestinal systems. Pharmacological activation of MOP not only modifies the transmis- sion and perception of nociceptive stimuli, but also a host of other effects, including reduced respiratory drive in response to increased levels

Yingxue Li', Mark R Lefever', Dhanasekaran Muthu', Jean M Bidlack2, Edward J Bilsky1 & Robin Polt*1

'Department of Chemistry &

Biochemistry, BIOS, The University of Arizona, Tucson, AZ 85721, USA

department of Pharmacology, University of Rochester Medical

Center, 601 Elmwood Avenue, Rochester, NY 14642, USA

'Department of Pharmacology. University of New England College of

Osteopathic Medicine, II Hill Beach Road, Biddeford, ME 04005. USA

*Author for correspondence:

Tel.: +1 520 621 6322 Fax:+I 520 621 8407

E-mail: [email protected]

FUTURE SCIENCE

10.4155/FMC.11.195© 2012 Future Science Ltd Future Med. Chem. (2012) 4(2), 205-226 ISSN 1756-8919 205

I Li, Lefever, Muthu, Bidlack, Bilsky & Polt

Figure 1. Opioid receptors. The p-opioid receptor G-protein-coupled receptor derived from bovine rhodopsin by homoiogy modeling [12].

of C02> opioid-induced bowel dysfunction, abuse liability and pruritus (itching). Repeated or prolonged MOP activation results in adap- tations that manifest as tolerance and physical dependence, further complicating management of chronic pain sufferers and patients with substance abuse disorders. Mixed opioid agonists/ antagonists and partial MOP agonists have been used to limit some of the opioid side effects with a mixed degree of success. Clearly, opioid pharmacology is a complex issue [14].

The DOP receptor [15] was originally characterized using the mouse vas deferens smooth muscle tissue preparation (mouse vas deferens [MVD] assay) [16]. The receptor is widely distributed anatomically, being found in the same general anatomical areas as MOP. DOP also contributes to a variety of physical and emo- tional effects, including initiation of movement [17], regulation of pain and reward circuitry, as well as other complex CNS behaviors (mood/ affect and anxiety). The issues of cellular co- localization of DOP and MOP, as well as the formation of functional 'heterodimers' con- tinue to be the subject of considerable interest [18-20]. Despite the progress that has been made in the localization of opioid receptors [21], the precise localization and neuronal and cellular pathways through which these three receptor

types work remains incompletely understood [22,23]. Although DOP receptors contribute to analgesia, euphoria and physical dependence, DOP agonists may be able to produce broad spectrum analgesic efficacy with reduced propensity to produce classic opioid side effects [24].

The KOP receptor, named after the K-agonist ketocyclazocine, is not as well studied as MOP and DOP, although it has attracted interest from pharmaceutical companies, as either a stand- alone drug [25,26] or in conjunction with agonism of other opioid receptors [27]. Stimulation of CNS KOP receptors is generally associated with dysphoria and psychomimetic effects, along with limited analgesic efficacy, especially in men [28]. Peripherally active KOP agonists produce antinociception in animal models of pain [29] and are being explored as targets for analgesia in several chronic pain states [18,30,31]. KOP antagonists are also being investigated as treatments for addiction and depression [32].

The diversity of the endogenous neuropeptides and their receptors provide many opportunities for drug discovery [33]. If there were a straightforward methodology for converting endogenous neuropeptides into useful CNS drugs, then a new and potentially sea-chang- ing pharmacopeia would be available for the treatment of CNS disorders.

Endogenous opioid peptide agonists Since the discovery of the two endogenous penta- peptides, Met-enkephalin and Leu-enkephalin in the 1970s, perhaps as many as 300 endogenous neuropeptides have been identified in widespread locations throughout the CNS TABLE I. Endogenous opioid peptides and their receptors undergo modulation in response to various physiological conditions, such as inflammation, tissue injury, pain and other Stressors [34].

The three classes of endogenous opioid peptides (enkephalins, endorphins or dynorphins) are typically assigned to the three types of opioid receptors (DOP, MOP and KOP, respec- tively). This approach is misleading since the absolute selectivity of each peptide is limited, and it neglects the fact that there are many cleavage variants of the neuropeptides, splice variants for the receptors and, likely, variations in their glycoforms. The endogenous peptides are not orthogonal and neuropeptide receptors might, for example, just as easily be thought of as 'metorphamide receptors' or 'enkephalin receptors' [35]. Moreover, a-endorphin and y-endorphin have been found to be inactive at

206 Future Med. Chem. (2012) 4(2) future science group I

Opioid glycopeptide analgesics derived from endogenous enkephalins & endorphins

Table 1. Endogenous opioid peptides'.

Peptide Sequence Subtype

Enkephalins

Leu-enkephalin YGGFL 8 receptor/p receptor

Met-enkephalin YGGFM p receptor/8 receptor

Metorphamide YGGFMRRV-NH2 8 receptor/p receptor

Peptide E YGGFMRRVGRPEWWMDYQKRYGGFL25 p receptor/K receptor

Endorphins

ß-endorphin YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE3] p receptor/8 receptor

y-endorphin YGGFMTSEKSQTPLVTL,7 p receptor/unknown

a-endorphin YGGFMTSEKSQTPLVT., p receptor/unknown

Dynorphins

Dynorphin A YGGFLRRIRPKLKWDNQ17 K receptor (p receptor)

Dynorphin B YGGFLRRQFKVVT)3 K receptor (p receptor, 8 receptor)

Dynorphin18 YGGFLRRQ8 K receptor (p receptor, 8 receptor)

a-neoendorphin YGGFLRKYPK K receptor (p receptor, 8 receptor)

ß-neoendorphin YGGFLRKYP K receptor (p receptor, 8 receptor)

Nociceptin/orphanin FQ

Nociceptin FGGFTGARKSARKLANQ 0RL1

Endomorphins

Endomorphin-1 YPWF-NH-, p receptor

Endomorphin-2 YPFF-NH2 p receptor

Dermal peptides

Dermorphin YaFGYPS-NH2 p receptor

Deltorphin A YmFHLMD 8 receptor

Deltorphin C YaFDVVG-NH2 8 receptor 'A few of the opioid peptides isolated from the mammalian CN5 and from the skin of amphibians are illustrated. Their common names, sequences and suggested receptor binding activities are shown. Weaker binding activities are in parentheses.

sites that are sensitive to ß-endorphin [36] and other sites have been found that are sensitive to y-endorphin, which have been referred to as 'non-opioid' in nature [37,38).

Schwyzer's membrane compartment concepts Schwyzer articulated critical roles for the membrane in peptide-receptor binding events (FIGURE 2) [39]. Although his 'membrane compartment theory' may have overstated the influ- ence of the membrane in differentiating receptor selectivity (u vs 8 vs K), it is clear that the membrane environment does play critical roles in pre-organizing the peptide conformation prior to binding, as well as the peptide-receptor binding event itself [40], We view binding as a three-step process:

■ Adsorption of the peptide ligand to the membrane. This promotes receptor binding by reducing a 3D search for the receptor to a faster 2D search. Surface-assisted 'reduction- of-dimensionality' calculations, performed by

Polya in 1921, were examined by Max Del- briick in which he quantitatively demonstrated the viability of this theory [41,42];

a Conformational changes in the peptide induced by the asymmetric environment of the membrane. Amphipathicity of the peptide is believed to reorganize the peptide-membrane aggregates into minimal energy states [43,44];

The binding event itself. GPCR binding and activation is a complex, multifaceted phenom- enon. It is, however, beyond the scope of this review.

The neurovascular unit & the BBB In order to enter the brain and CNS, pharmaceutical agents must first penetrate the BBB (FIGURE 3). There are several strategies available to make a peptide metabolically stable but the transport of a peptide across BBB is still remains a major hurdle in developing CNS drugs [45]. Contrary to the belief that small molecules with molecular weights under 400 readily cross the

I future science group www.future-science.com 207

REVIEW Li, Lefever, Muthu, Bidlack, Bilsky & Polt

Pre-synaptic membrane Release

Post-synaptic membrane

Sn

^o

:Q-

Membrane hopping

Active conformation

:Q Inactive

conformation

u receptor

Figure 2. Membrane hopping. Endogenous opioids associate with the membranes (kon » k )f) and bind to one or more of the opioid receptors via a membrane-bound conformation (Fisher's lock and key). Studies show that active (folded) conformations are favored in the membrane and inactive (random coil) conformations are favored in the absence of a membrane. Incorporation of glycosides, represented by the 270° arc, can shift the kon/ko(f equilibrium to facilitate 'membrane hopping'.

Figure 3. The neurovascular unit. The neurovascular unit forms the blood-brain barrier that prevents the passage of most peptides and other polar substances from the capillaries into the brain.

BBB, it has been observed that almost 98% of small molecules do not readily cross the BBB. Further, evidence has accumulated that peptides can penetrate the CNS by several different mechanisms [46-49]. These observations clearly indicate molecule size is not the primary factor, but rather the overall physiochemical characteristics of the molecule is critical for BBB transport. Since the BBB is composed of endothelial membranes, many research groups focus on designing peptides with increased lipid solubility. Simply increasing the lipid solubility of a drug molecule may have undesirable effects, such as decreasing solubility and bioavailability, and increasing plasma protein binding.

The BBB is composed of endothelial membranes that function as a continuous lipid barrier that protects the brain from toxic substances by preventing their entrance from bloodside to CNS. The BBB is also an enzymatic barrier that poses additional challenges in developing peptide/protein-based CNS drugs. Further, it has been viewed recently as a regulatory interface between the CNS and circulation with nutritional, homeostatic and communication functions. Understanding of the principles and physiology of BBB has improved a great deal in the past decade. Moreover, evidence is accu- mulating that many peptides and proteins cross the BBB in amounts sufficient to affect CNS function. It is now clear that the BBB is not an absolute physical barrier but a regulatory tool that controls the delivery of the substances to the CNS. Strategies based on this principle are proving to be very successful [so]. Other strategies using 'molecular umbrellas' [51], 'Trojan horses' [52] and BBB 'shuttles' [53] have been proposed by various groups. We have successfully applied glycosylation as a strategy to improve the BBB penetration, as well as the stability and systemic availability of enkephalins and the larger endorphin-like peptides.

Glycopeptide synthesis

Initially, O-linked glycopeptides were considered to be exotic substances, and were difficult to evaluate as drugs simply because the synthetic methods required to produce them in tangible amounts were lacking. In the last 40 years this situation has changed, however, glycopeptides are still significantly more difficult to produce than simple peptides, even if the requisite Fmoc-amino acid glycosides are commercially available, and particularly so if the glycosides require synthesis [54.55]. Enzymatic approaches

208 Future Mod. Chem. (2012) 4(2) future science group I

Opioid glycopeptide analgesics derived from endogenous enkephalins & endorphins REVIEW

have been incorporated into chemical methods for serine and threonine glycosides [56]. The ideal methodology should produce high yields of pure diastereomers, typically ß-anomers for the highest stability. Many classical methods produce high yields of the desired anomers, but require the production of labile glycosyl donors or very reactive (e.g., unstable) promoters [57].

Production of O-linked glucosides and lac- tosides of enkephalins and endorphins requires the corresponding acetate-protected glycosides of Fmoc-serine or Fmoc-threonine. The benzo- phenone Schiff base appeared to be a very good protection for amino groups of serine and threonine for glycoside formation [58]. O-glycosides of these amino acids with different monosaccharides, aminosugars and deoxysugars were obtained with excellent yield and very high stereoselectivity [59]. Glycoside peracetates have been used as building blocks for solid-phase glycopeptide synthesis, and significant improve- ments have been made in the production of the glycoside building blocks [60]. An even more efficient and direct approach to these precur- sors has now been developed that proceeds directly from Fmoc-serine or Fmoc-threonine as glycosyl acceptors, and either ß-D-glucose peracetate or ß-lactose peracetate in the presence of 'minimally competent' Lewis acids, such as indium(HI)bromide (FIGURE 4) [61].

The glycopeptides can be synthesized manually based on established solid-phase yV-fluorenylmethoxycarbonyl methods (Fmoc chemistry) (FIGURE 5). The side chain-protected amino acids used by our research group were Fmoc-Lys(Boc)-OH, Fmoc-Glu(OfBu)-OH, Fmoc-Asn(Trr)-OH, Fmoc-D-Thr(Buf)-OH, and Fmoc—Tyr(Bur)-OH. For support we have used Rink amide MBHA 1% DVB resin, with substitution typically ranging from 0.2- 0.8 meq/g. Best results (e.g., highest purities of the crude glycopeptides) were obtained by coupling well below the resin capacity and then capping the excess capacity with acetic anyhydride (Ac20). Coupling of the Fmoc-amino acids was achieved using manual coupling methods with or without microwave heating or, typically, using mechanization, with extended reaction times or heating only for coupling of the glycosidic residue and the residue following, which may be regarded as an extremely hindered case of amino acid coupling. Use of NMP as a solvent rather than the more polar DMF also aided these couplings. Manual coupling reactions and critical couplings performed during mechanized

-f M = Sc, B; strong Lewis acids

£^Y -OAc MX, OAc z$

OAc

X-M-OAc xe

ROH Aer OAc

X-M-OAc Je

M = In -HOAc j

Minimally competent Lewis acids

Figure 4. Minimally competent Lewis acids as glycosidation promotors. Lewis acids such as lnBr3 can dissociate (lower pathway) from the displaced acetate to form acetic acid and regenerate the Lewis acid catalyst. Stronger Lewis acids remain associated with the acetate (upper pathway) to produce a Bransted acid and, generally, require a full equivalent of the Lewis acid.

coupling were typically monitored using Kaiser's ninhydrin test.

The Fmoc group was removed from the A-terminus of the growing glycopeptide chain using a mixture of 3% piperidine and 2% diaza- l,3-bicyclo[5.4.0]-undecane in DMF for 10 min with argon bubbling as agitation. The ace- tyl protecting groups of the glycosides and the N-terminal Fmoc could be removed with 80% hydrazine hydrate (H2NNH2»H20) in CH^OH with argon agitation 3X for approximately 2 h. A superior method used Boc protection for the last amino acid, which survived the hydrazine treatment but was removed by TFA. The synthetic glycopeptides were cleaved from the Rink resin with a 'TFA cocktail', F3CCOOH:Et3Si

H:H20:PhOCH3:CH2Cl2 (8:0.5:0.5:0.05:1), which also removed the side chain protection, and the N-terminal Boc group, if that method is employed. The crude glycopeptides were precipitated in cold diethylether, redissolved in H20 and then lyophilized prior to reverse- phase HPLC. A preparative scale Clg column (Phenomenex® 250 x 22 mm, 250 x 55 mm or equivalent) was used, with an acetonitrile-water (CH3CN-H20) gradient containing 0.1% TFA to obtain glycopeptides of greater than 97% purity. Homogeneity of the pure glycopeptides was confirmed by analytical reverse-phase HPLC and MS.

Glycopeptide analgesics based on enkephalins Our initial attempts at improving the CNS bioavailability of opioid peptides came from collaborations between the Chemistry and Pharmacology Departments at the University of Arizona led by Victor Hruby [62]. In 1983

I future science group www.Tuture-science.com 209

Li, Lefever, Muthu, Bidlack, Bilsky & Polt

CH,0 NH-Fmoc

H,CO'

Rink amide MBHA resin

S Fmoc-NH-Rink (3

1) 3% Piperidine/2% DBU/DMF Repeat)

2) Fmoc-Aaa-OH/DIC/HOBt v_^ (last amino acid is Boc-protected one)

Boc-Tyr(tBu)-D-Thr(tBu)-Gly-Phe-Leu-Xxx-Asn(tTr)-Leu-Xxx-Glu(OSu)-Lys(Boc)-Xxx-Leu-Lys(Boc)-Ser[0--b-Glc-(OAc)J-Leu-NH-Rink—{~k

3) H2NNH2-H20/MeOH (8:2)

Boc-Tyr(IBu)-D-Thr(Su)-Gly-Phe-Leu-Xxx-Asn(rTr)-Leu-Xxx-Glu(Ofiu)-Lys(Boc)-Xxx-Leu-Lys(Boc)-Ser[0--b-Glc-(OH)J-Leu-NH-Rink—TJ

4) TFA/Anisole/Et3SH/H20/CH2CI2

.0 H2N-Tyr-D-Thr-Gly-Phe-Leu-Xxx-Asn-Leu-Xxx-Glu-Lys-Xxx-Leu-Lys-Ser[0-b-Glc-(0H)J-Leu-4

NH2 Xxx ■ Aib, Ala, Gly, for example

Figure 5. Glycopeptide assembly. MBHA-functionalized Rink polystyrene resin was used to provide the C-terminal amides upon cleavage after classical Fmoc construction of the glycopeptides. Treatment with hydrazine hydrate (H2NNH2«H20) in methanol (CH3OH) was required to remove the acetates from the glycoside moiety prior to cleavage from the Rink resin. A Boc-protected amino acid may be used for the final amino acid (O-tBu-Tyr), which is cleaved with the TFA cocktail.

this group successfully synthesized and characterized a series of cyclic penicillamine containing enkephalin analogues (e.g., DPDPE) that had higher affinity and selectivity for DOP [63]. Incorporation of an unnatural amino acid (D-penicillamine) and the cyclic constraint into the peptide enhanced both its stability and DOP selectivity. One hypothesis was that by increasing the lipophilicity of an already quite lipophilic DPDPE, BBB penetration could be increased, which was confirmed in an in vitro BBB model that used bovine brain microvessel endothelial cells [64].

Concurrently, we tested an alternative hypothesis, which, in retrospect, was naive and incorrect, whereby attachment of a glucose molecule to the modified enkephalin peptide would make the overall ligand a substrate for the Glut-1 transporter [65]. In an effort to enhance CNS bioavailability, we synthesized a series of enkephalin analogues (TABLES 2-4). It was predicted that CNS delivery of the enkephalin molecule across the BBB would be increased, and that we would observe antinociceptive activity following systemic administration. While the Glut-1 hypothesis eventually proved to be incorrect, the enkephalin glycosides did penetrate the BBB very effectively and produced potent and long- lasting antinociception in mice after intravenous (iv.) or intraperitoneal (ip.) injection [66].

TABLE 2 highlights some of the initial enkephalin glycopeptides that were synthesized [67]. Glycoside placement proved critical for affinity as determined by radioligand binding studies, and efficacy, as determined by GPI and MVD assays. Glycosylation sites close to the N-terminus resulted in reduced affinity for both DOP and MOP. Extension of the modified enkephalin peptide at the C-terminus allowed for glycosylation while preserving opioid receptor affinity, with some compounds retaining moderate DOP selectivity while others had approximately equal affinity for DOP and MOP.

Two glycopeptides, ß-glucoside 3 and 4 (TABLE 2) were tested in mice for their ability to produce CNS-mediated antinociception after systemic administration, and were compared with the unglycosylated peptide control I. Both glycopeptides 3 and 4 produced dose- and time- related antinociception following ip. injection into mice, whereas the unglycosylated control peptides did not produce any measurable effects. The primary obstacles for better characterization of these glycopeptides were the somewhat tedious synthesis of the cyclic disulfides and the relatively low potency of the compounds (-30 mg/kgA5l) values).

Larger quantities of a linear enkephalin glycoside based on Roques's so-called 'delta-enkephalin or DTLET (YtGFLT,

210 Future Med. Chem. (2012) 4(2) future science sroup I

Opioid glycopeptide analgesics derived from endogenous enkephalins & endorphins | IAEVIEW

Table 2. Cyclic disulfides related to DPDPE*.

Compound Structure IC50(nM)

8 receptor u receptor MVD GPI

1 pj 8-, o 6.1-6.4 30-43 5.5 26 Tyr-D-Cys-Gly-Phe-D-Cys-Ser—U-NH2

HO-I

2 - 3900 7700 520 3700 r° ° i o Tyr-D-Cys-Ser-Phe-D-Cys-Gly—U-NH,,

ß-D-GIc-CM

3 HS-, pSH 0 9.9 42 24 110 Tyr-D-Cys-Gly-Phe-D-Cys-Ser-U-NH2

ß-D-Glc-0-l

i s s~i o 26-46 45-53 13 60 Tyr-D-Cys-Gly-Phe-D-Cys-Ser-U-NH2

ß-D-Glc-0-l

5 i-s—s-f o 85 48,000 560 40,000

Tyr-D-Pen-Gly-Phe-D-Pen-Ser-U-NH2

ß-D-Glc-0-l

19 6 rS S-, o 32 Tyr-D-Cys-Gly-Phe-D-Cys-Ser-U-NHj

ß-D-Xyl-0-l

pS S-| o 10 53.3 Tyr-D-Cys-Gly-Phe-D-Cys-Ser-"-NH2

a-D-Glc-oJ

48 9 is sn p Tyr-D-Cys-Gly-Phe-D-Cys-D-Ser—U-NH2

/o-ß-D-GIC

'Study of a series of cyclic disu/fide glycosides of enkephalin showed that placement of the glycoside was critical to maintain opioid activity and that 5 receptor selectivity was reduced in this series of compounds. GPI: Guinea pig ileum; MVD: Mouse vas deferens.

Tyr-D-Thr-Gly-Phe-Leu-Thr) were produced. The parent unglycosylated peptides, 9 and 10, retained high affinity for both DOP and MOP, relatively weak binding to KOP, and displayed a slight preference (-tenfold) for functional activity in the mouse MVD assay versus GPI assay [68]. Both compounds were extremely potent (<0.1 nmol A^0 values) following intracerebroventricular (i.c.v.) administration but required very large doses iv. to produce any antinociception in the mouse 55°C tail-flick. The addition of a glucoside to a serine in the sixth position of the peptide resulted in retention of modest selectivity for DOP over MOP in functional MVD/ GPI tissue assays and in receptor-binding studies (69]. Both the parent peptide (9) and glycosylated analogue (12) were extremely potent in the mouse tail-flick assay following intracerebroventricular injection. However, glycopeptide 12 was significantly more potent following systemic routes of administration (iv., ip. and sc). In situ BBB studies in rats also indicated, despite the increase in MW and increased water

solubility, that the glycopeptide penetrated the BBB more effectively than its unglycosylated peptide counterpart 9 [70]. When compared with morphine, glycopeptide 12 resulted in lower levels of physical dependence as indicated by naloxone-precipitated withdrawal.

In an effort to further explore the structure- activity of glycosylation [71], a number of glycopeptides were synthesized to determine if the type of monosaccharide altered the transport characteristics and systemic potency of the lead peptide pharmacophore; if di- or tri-saccharides provided any additional benefit to pharmacokinetic and pharmacodynamic properties; and if bis- or tris-monosaccharides were viable alternative strategies for improving BBB transport and systemic potency [72]. In addition, several other modifications were made to explore the geometry of the attachment point (D VS L amino acid) of the glycoside and to see if the more ste- rically hindered threonine attachment diffeted from serine in its effects on activity. It should be noted that we stayed with the linear enkephalin

I future science sroup www.future-science.com 211


Table 3. Linear glycopeptlde amides as 8 receptor/a receptor opiold agonists.

Compound Structure K (nM) A 0

5 receptor M receptor K receptor Intracerebro- ventricular (nmol)

Intravenous (umol/kg)

Morphine CH3-N

/T^X/\\ -1/2 H2SO„

HO Or' OH

290 ±38 0.79 ±0.12 12.0 ± 1.3 2384 7.84

9 0 Tyr-o-Thr-Gly-Phe-Leu-Ser-U-NH,

HoJ

4.1 ±41 1.4 ±0.08 34.0 ±2.2 0.068

0.038

46.4

32.6 10 0 Tyr-D-Thr-Gly-Phe-Leu-Thr-U-NH2

HCM

9.71 11.7 ND

II 0 Tyr-D-Thr-Gly-Phe-Leu-Ser-U-NHj

ß-D-Xyl-0-l

46.0 65.8 ND 0.092 9.45

12 0 Tyr-o-Thr-Gly-Phe-Leu-Ser-U-NH2

ß-D-Glc-0-l

7.0 ± 1.2 2.4 ±0.017 49 ±4.3 0.023 11.4

13 0 Tyr-D-Thr-Gly-Phe-Leu-Ser-U-NH2

a-D-Man-O-'

0 Tyr-D-Thr-Gly-Phe-Leu-Thr-U-NH2

P-D-GIC-OJ

0 Tyr-D-Thr-Gly-Phe-Leu-D-Ser—U-NH2

^O-ß-D-GIc

23.0 15.2 ND 0.033 16.7

14 16.8 39.8 ND ND 0.022

15 54.4 297.8 ND 0.035 ND

16 31.8 ND 0 Tyr-D-Thr-Gly-Phe-Leu-D-Thr—U-NH2

24.5 0.040 22.0

i-O-p-D-GIc

Glycopeptide monosaccharides based on DOP-preferring enkepha ND: Not determined.

nd DsGFLT (DSLFT). in sequences developed by BP Roques, DtGFLS (DTLES) a

parent peptide as it had roughly equal affinity for DOP and MOP receptors. This was important in assessing potential effects of glycosylation on preferential biasing for DOP or MOP.

The initial Roques-based linear peptides tested had either an L-Ser or L-Thr added to the sixth position of the peptide. The geometry of the glucoside attachment did not impact functional potency/efficacy in the in vitro or in vivo assays (L-Ser vs D-Ser or L-Thr vs D-Thr). For the monosaccharides, the ß-xylose was approximately two-times more potent than ß-glucose or a-mannose following iv. administration. The three disaccharides (ß-lactose, ß-maltose and ß-melibiose) were all more potent than the best monosaccharide tested, with the ß-melibioside being the most potent of the three.

Based on these results, we synthesized additional glycopeptides that incorporated a trisaccharide (ß-maltotriose) to see if additional size/

bulk of the carbohydrate moiety would lead to further increases in iv. potency. The experimental data indicated a modest fall off in binding affinity and potency in the in vitro and in vivo functional assays. We also extended the hexa- peptide to include one to two additional Ser or Thr attachment points with ß-glucose (bis- and tris-monosaccharides) to more fully explore the structure—activity relationship. In all cases, the additional glycosyl bulk reduced potency following i.c.v. administration, and the one compound tested iv. was significantly less potent than the original glycopeptide (ß-glucoside and L-serine attachment).

Additional studies confirmed aspects of the in vivo studies [67]. Larger carbohydrates reduced octanohsaline partitioning (logD value), indi- cating greater water solubility (parent peptide < monosaccharide < disaccharide < trisccharide). Serum and brain stability of the glycopeptides


Opioid glycopeptide analgesics derived from endogenous enkephalins & endorphins | REVIEW

Table 4. Linear glycopeptide amides as p receptor/< i receptor opioid agonists.

Compound Structure K, (nM) A 50

5 receptor p receptor K receptor Intracerebro- ventricular (nmol)

Intravenous (pmol/kg)

Morphine CH3-N

// %£f~\ • 1 /2 H2S04

290 ± 38 0.79 ±0.12 12.0 ±1.3 2384 7.84

17

H0' ^0° T3H

0 Tyr-D-Thr-Gly-Phe-Leu-Ser-U-NH2

ß-D-Gal-0-(1 -4)-ß-D-GIC-0 J

9.20 ± 1.70 42.0 ±5.0 0.018 3.20 5.00 ±0.65

6.48 41.9 ND 0.034 2.16 18 0 Tyr-D-Thr-Gly-Phe-Leu-Ser-U-NH2

a-D-Glc-0-(1 -6)-ß-D-Glo-0-l

19 31.0 ±0.3 ND ND 0 Tyr-D-Thr-Gly-Phe-Leu-Ser-U-NH2

ß-D-GIC-0-(1-4)-ß-D-Glc-0 J

0 Tyr-D-Thr-Gly-Phe-Leu-Ser-U-NH2

a-D-Glc-0-(1 -4)-ß-D-Glc-0 J

13.0 + 0.55 4.5 ±0.12

20 9.86 30.8 6.82 ND 0.062


a-D-Glc-0-(1-4)-a-D-Glc-0-(1-4)-ß-o-Glc-0-'

25.0 56.7 ND 0.061 10.9

22 0 Tyr-D-Thr-Gly-Phe-Leu-Ser-Ser—U-NH„

II ß-D-GIc-O-1 L-O-ß-D-GIc

ND ND ND 0.380 140.8


9 PhCH2-0-P-0—'

3.3 ±0.33 8.7 ±0.93 130 ±6.2 0.093 30% at 32.0 mg/kg

24

HO

3.9 ±0.19 310 ±66 0.34 54.1 0 Tyr-D-Thr-Gly-Phe-Leu-Ser-U-NH2

l ! HO-P-0—'

7.9 ± 0.73

25

HO

0 14.0 ±0.34 1100 ± 13 3% at 0.22 5.63 Tyr-D-Ala-Phe-Glu-Nle-Nle-Thr-U-NH., 10.0 uM

ß-D-Glc-0 —'

Glycopeptide disaccharides, and phosphates based on 8 receptor-preferrinc ND: Not determined.

enkephalin seqi tGFLS (DTLES) and D sGFLT (DSLET). ences developed by BP Roques, D

also increased with these substitutions. In an in situ model of BBB transport the disaccharide proved to be the most readily transported with the trisaccharide having a reduced RBr value (though still superior to the unglycosylated control). We extended the in vivo work by adding additional pain assays to assess efficacy. The disaccharide 17 produced potent antinociception in the formic acid, acetic acid and carrageenan assays following systemic administration (all of these pain assays have an inflammatory component).

Based on this modest library of glycopeptides, we chose the ß-lactoside (17) as the lead molecule

to pursue more advanced in vivo characterization. While not the most potent of the disaccharides, the compound was much easier and less costly to synthesize compared with melibiose. Glycopeptide 17 also had some additional desir- able characteristics, including being highly water soluble (>50 mg/ml). Based on these findings, we advanced 17 into a more complete characterization of its antinociceptive efficacy and side-effect profile.

As expected, 17 produced full efficacy in the GTPyS assay with a modest selectivity for DOP over MOP. This profile was similar to what was


REVIEW | Li, Lefever, Muthu, Bidlack, Bilsky & Polt

1.6

1.2

0.8

0.4

®

1- 0.8 0.9 0.65 0.7 0.75 0.8 0.85 0.9 0.95 3.5 3 2.5 2 1.5 1 0.5 0

Amphipathicity (Connolly-derived) Hydrodynamic volume (g.u)

Figure 6. Antinociception studies indicate a U-shaped or V-shaped curve when the A50 potency values are correlated with predicted amphipathicity. The hydrodynamic values (glucose units) or Connolly-derived amphipathicity values are plotted along the X-axes, and A50 values derived from mouse intravenous tail-flick data are plotted on the Y-axis. Both analyses produce a U-shape or V-shape, as predicted by the biousian hypothesis [84]. The amphipathicity values were calculated using the formula A = e-Awa,e,Mlipid, where Awater _ tne Connolly surface area of the hydrophilic moiety (Ä2) and Alip,d = the Connolly surface area of the rest of the lipophilic peptide message segment YaG(N-MeF).

observed in the functional MVD and GPI tissue assays. We confirmed this profile in vivo by pretreating mice with various opioid antagonists. The general antagonist naloxone completely blocked the actions of 17 in the 55°C tail-flick assay in mice. In contrast, the peripherally selective antagonist naloxone methiodide did not alter the agonist actions of the compound. Subtype selective DOP (naltrindole) and MOP (ß-FNA) antagonists each partially blocked the antinociceptive actions of 17 and, when com- bined, they completely eliminated the agonist actions. The KOP-selective antagonist nor-BNI was without effect.

As a lead molecule, 17 was also tested in several rat models of pain to determine how broad an antinociceptive spectrum the compound might have. The first assay used was a post- surgical incision model of the hind paw [73]. Morphine and 17 both produced dose-related reversal of the tactile allodynia associated with the injury [DGluvELlSfr^i.,UNPUBLISHED DATA]. On a umol/kg basis, 17 was almost equal to morphine in terms of potency. Similar results were seen in a subchronic inflammatory pain model induced by complete Freund's adjuvant, although, in this case, 17 had greater potency than morphine, possibly due to enhanced DOP signaling under inflammatory conditions. Finally, 17 was compared with gabapentin in a rat spinal nerve ligation model of neuropathic pain. Glycopeptide 17

produced potent reversal of the tactile allodynia and thermal hyperalgesia post-ligation, whereas gabapentin only reversed tactile allodynia at the doses examined [DGIUVELISET.«!.., UNPUBLISHED

DATA] . Collectively, the data indicate that a mixed DOP/MOP agonist has a broad spectrum of antinociceptive effects in acute and chronic pain models, including ones that have inflammatory and/or neuropathic pain components.

One of the initial screens for side effect was to inject increasing doses of 17 or morphine and col- lect locomotor data in an automated open field assay. Morphine and other MOP agonists stimu- late forward locomotion in imprinting control region mice. This effect becomes pronounced at near maximal and supramaximal antinociceptive doses. The mixed MOP/DOP agonist 17 produced an initial and transient decrease in forward locomotion that was replaced by a very mild stimulation of activity at later time points. We further investigated the initial inhibition of locomotor activity by pretreating mice with naloxone methiodide or nor-BNI. Both pretreat- ments attenuated the effects of 17 on locomotion and completely eliminated both effects when the two opioid antagonists were administered simultaneously. This indicated that stimulation of peripheral opioid receptors can produce a transient decrease in exploratory locomotor behavior and there may be a modest K-agonist effect of the compound in the CNS that contributes to reduced stimulation of locomotor activity but does not contribute to the antinociceptive effects in the 55°C tail-flick assay.

Interestingly, one of the other gross observable differences between 17 and morphine is a lack of Straub tail and muscular rigidity with 17. We quantified this effect in dose—response curves versus antinociception with both morphine and the mixed agonist 17. The potential of morphine to produce both effects overlapped, whereas it took much higher doses of 17 to produce the muscular rigidity and Straub tail compared with its antinociceptive effects.

Based on the mixed DOP/MOP profile of 17, we were interested in evaluating the tolerance and physical dependence liability of the compound relative to morphine. For tolerance studies, we used a common paradigm involving twice-daily injections of the approximate A90

doses of the agonist (or vehicle) for 3 days. On the morning of day 4, full dose-response curves were constructed for each compound in the agonist- and vehicle-treated animals. Repeated doses of morphine resulted in an approximately

214 Future Med. Chem. (2012) 4(2) future science sroup I

Opioid glycopeptide analgesics derived from endogenous enkephalins & endorphins j REVIEW

Table 5. Glycopeptides based on DAMGO. >

Compound Structure K. (nM) A

Intracerebro- ventricular (nmol)

50


5 receptor preceptor K receptor

Morphine CH,-N

f^V/A • 112 H.SO,

290 ±38 0.79 ±0.12 12.0 ± 1.3 2384 7.84

DAMGO

HO O1 OH

0 H 990 ±35 0.56 ± 270 ±9.3 30 1.88

26

Tyr-o-Ala-Gly-N-Me-Phe "- N-CH;CHzOH

0

0.006

0.20 600 ± 44 0.68 ±0.02 190 ±9.3 2.0

27

Tyr-D-Ala-Gly-N-Me-Phe-Ser -U-NH,

HO-I

2.0 0.27 0 730 ±66 1.30 ±0.16 160 ± 10 Tyr-D-Ala-Gly-N-Me-Phe-Ser -U-NH2

ß-D-Xyl-0 -1

28 0

Tyr-D-Ala-Gly-N-Me-Phe-Ser ^-NH2

P-D-Glc-O-'

54% at 10 uM

1.30 ±0.14 270 ±2.5 19 0.72

29 0

Tyr-D-Ala-Gly-N-Me-Phe-Ser ■"-NH2

ß-D-Gal-0-(1-4)-ß-D-Glc-0 -I

1600±129 0.66 ±0.05 350 ±51 2.0 1.15

30 4.2 ±0.34 570 ± 9.8 33 30% at 0 55 ±4.6 Tyr-D-Ala-Gly-N-Me-Phe-Ser -"-NK,

0 ] II

PhCH2-0-P-0—I

38.1 mg/kg

31

HO

0 1400 ±180 42 1.5 3.8 ±0.73 31% at Tyr-D-Ala-Gly-W-Me-Phe-Ser ■"-NH,

0 I II

HO-P-0—I

10 uM

HO

Glycopeptide disaccharides and phosphates based on the p receptor-preferring sequences

13-fold rightward shift in the A-0 value indicat- ing substantial development of antinociceptive tolerance. Equivalent doses of 17 (in terms of analgesia) resulted in a significantly reduced rightward shift (<fivefold). Morphine and glycopeptide 17 have similar durations of action and AUC values, making the comparisons more straightforward.

For assessment of physical dependence liability, we used both an acute (single high- dose administration of agonist) and chronic (twice-daily injections for 3 days) dependence protocol. In both cases, injection of the general opioid antagonist naloxone was used to precipitate withdrawal and several indices of withdrawal were recorded (vertical jumps and paw tremors, for example). The level of physical dependence/severity of withdrawal was con- sistently lower with the 17 exposure compared

with equivalent exposures of morphine [74]. The working hypothesis for explaining these results is that the antinociceptive effects of the mixed DOP/MOP compound synergize at the cellular or network level, whereas the processes that drive tolerance and/or physical dependence are additive or subadditive. A predominantly MOP- selective agonist, on the other hand, requires significant occupation of the MOP receptors at sites both responsible for antinociception and tolerance/dependence. Other explanations are possible, including the formation of heterodimers with the glycopeptide (17) versus the small molecule (morphine) that lead to activation of different signaling pathways.

To further characterize the side-effect profiles of the glycopeptide, two commonly used assays for assessing MOP effects were used (gastrointestinal transit and respiratory depression). We



® Pro

Val Thr

Lys Phe

Asn Ala

lie He

Asn Lys

fö ^

Lys Lys

Leu Pro

Thr

Asn Lys

Phe

®

Lys

Lys

lie

Leu.

Ala

Pro13 lie22

/~N Ala Asn

Lys

[' ~V Lvs21

Lys24 Val15 y Ash

ß-endorphin, 13-31

Figure 7. Representations of the amphipathic helical region in ß-endorphin. (A) Human ß-endorphin [12-28] represented both as an a-helical net projection (left) and as a n-helical net projection (right) [90]. The lipophilic (hydrophobic) residues are circled. (B) Human ß-endorphin [12-30] represented as an axial projection of a it-helix [«].

had predicted that the DOP/MOP profile would have a reduced effect on these parameters compared with equivalent doses of morphine. This was not the case. Glycopeptide 17 also inhibited upper-gastrointestinal transit and suppressed respiratory response to elevations in CO, on the minute ventilation parameter. The former may have been due to the apparent higher concentrations of glycopeptide 17 in the peripheral circulation compared with CNS, thus, overwhelming the MOP populations in the enteric nervous system. These values were estimated from the i.c.v. versus iv. potency ratios to produce antinociception for 17 versus morphine (more for- mal pharmacokinetic measures are currently being conducted). The respiratory depression

observations indicate that a slight preference for DOP over MOP is not sufficient to differentiate from a MOP selective agonist.

Additional studies were conducted with 17 with respect to its abuse liability. As mentioned previously, the level of locomotor stimulation with 17 was markedly reduced compared with morphine. The stimulation of forward locomotion is generally interpreted as an activation of mesolimbic dopamine systems and an indica- tor of abuse liability. Our group has also conducted preliminary studies using conditioned place preference and iv. drug self administration in rodents. In the conditioned place preference studies, morphine produced a significant place preference whereas antinociceptive equivalent

Figure 8. Micelle-bound structures of glycopeptide analogues determined by NMR related to (A) enkephalins and (B) endorphins [92,93].


Opioid glycopeptide analgesics derived from endogenous enkephalins & endorphins IAEVIEW

Table 6. The amphipathic helix address'.

Compound Message sequence

Link Helical address sequence 8 receptor

K.(nM)

u receptor K receptor

A


so


32 YtFGL P NLBEKALKSL-NH2 9.6

3.6

12.0

16

39

29

4.2

4.3

2.3 2.8 0.57 0.32 0.36 1.06 (» 10)

33

34

35

36

YtFGL

YtFGL

P

P

NLBEKALKS*L-NH2

NLBEKALKS**L-NH2

SL-NH2

S*L-NH2

NLBEKALKSL-NH2

NLBEKALKS*L-NH2

NLBEKALKS**L-NH2

2.6

8.2

2.3

5.7

0.58

0.11

0.064 YtFGL

YtFGL

GABA

GABA

GABA

GABA

11

16

31

38

56 0.059

0.143

0.046

0.165

2.29 1.12 0.28 1.13 1.41

carbohydrate (35 region (e.g., 38^

37

38

39

40

'For endorphin vs 26) clearly sh provides the be S = OH; S' = Gl DAVA: Delta an

YtFGL

YtFGL

YtFGL

49

11

11

4.3

0.97

37 cts on bi ansport,

GABA

YtFGL DAVA NLBEKALKSL-NH2 32 analogues 32, 33 and 34, the glycosylation state showed only minor effe ows a dramatic effect on intravenous potency, presumably by affecting tr t delivery,

c; S" = Lact. ino valeric acid; GABA: Gamma amino butyric acid; P: Proline.

64 0.162 ency The addition of c atures into the addres.

nding and intravenous pot and combining the two fe

doses of 17 did not (similar to vehicle). In rat self-administration studies 17 maintained significantly lower numbers of infusions than the more MOP selective agonists morphine and fentanyl. In addition, the cumulative latency to delivery of the first three infusions of 17 (at the peak of the dose-effect curve) was significantly longer than morphine and fentanyl [STEVENSON

ET AL. MANUSCRIPT IN PREPARATION]. These experiments suggest that the reinforcing effects of 17 are less than the prototypical MOP agonists morphine and fentanyl. The reinforcing effects of 17 were also evaluated in rhesus monkeys [75]. Under the conditions examined 17 did not support self-administration in rhesus across a series of doses/concentrations, although the results are more difficult to interpret due to species differences with respect to pharmacokinetics.

Current studies with DOP-selective peptides With respect to our analgesic drug-development efforts, the prior work with 17 (mixed DOP/ MOP agonist) indicated to us that greater DOP selectivity might be needed in order to differentiate a lead candidate from currently available MOP analgesics. This was based not only on the extensive characterization we had done with 17, but also the literature demonstrating involvement of DOP receptors in neuropathic and other chronic pain states, and the further improvement in side-effect profiles [76-78]. We have also been interested in exploiting potential differences between the small-molecule DOP agonists, such as BW373U86 and SNC80, and

the larger peptide-based deltorphin II analogues [79-82]. The former are ineffective in acute nociceptive assays that have high stimulus intensities, whereas the later are effective. This may be due to differences in ligand/receptor biasing or interactions with unique homo- or hetero-dimers of the DOP [83].

A series of glycosylated deltorphin analogues were synthesized that retained their high affinity, selectivity and efficacy at the DOP. Through an in vitro screening process, two to three lead compounds emerged, including glycopeptide

Opioid message^ Random coil

Opioid message Helical address Sugar

Opioid GPCR u, K0r8

Figure 9. Biousian behavior in a helix context. Modulation of amphipathic helix stability should modulate interactions with biological membranes and 'searching' for the receptor.

> future science sroup www.future-science.com 217

REVIEW | U, Lefever, Muthu, Bidlack, Bilsky & Polt

25. (TABLE 4) This compound exhibits a low nM EC value in the GTPyS assay and is very potent when injected i.c.v. (<1 nmol A,(| value) (KITSOS

£7-^L.MAUSCRIPT IN PREPARATION). GlyCOpeptide 25 is

also systemically bioavailable following iv. and p.o. dosing, although the oral dose requires a proprietary co-formulation technology developed by Unigene. We are currently conducting further assessments of efficacy and side effects of glycopeptide 25 and comparing them to morphine and to 17. These preliminary data suggest that we the efficacy of the 5-agonist 25 is at least

equal to the mixed u/S-agonist 17 with a side- effect profile on gastrointestinal transit that is superior to morphine and 17.

DAMGO-based MOP agonists To further explore and exploit the biousian hypothesis [84], the classical p-agonist (K.: 0.53 nM) DAMGO [85,86] was used as a lipophilic peptide message [87], and additional moieties added to provide a water soluble address to produce a series of MOP-selective ligands. Their pharmacology was assessed in vitro and in vivo

(A) Peplides in SDS

30,000

20,000

o E p

01 <a 3

s v

5

10,000

0 -

-10,000

■20,000

(B) Peptides in TFE

30,000 T 1

b 20,000 "

o E 5 10,000 -

.9- 0 - Ö) o 3

■o

'g -10,000 c 3 B

-20,000

190 200 210 220 230 240 250

Wavelength (nm) 190 200 210 220 230 240 250

Wavelength (nm)

@ Peptides in water

0 -.

-25,000 190 200 210 220 230 240 250

Wavelength (nm)

Figure 10. Circular dichroism to measure helicity. Modulation of the amphipathic helix stability can be directly measured by examining the circular dichroism behavior in (A) sodium dodecylsulfate, (B) trifluoroethanol and (C) water, and measuring the elipticity at 222 nm.

218 future Med. Chem. (2012) 4(2) future science group I


[88,89]. It was believed that the exploration of the biousian hypothesis [84] within the context of a pure MOP agonist would simplify inter- pretation of the results (FIGURE 6). The binding and antinociceptive effects of 26-29 are shown in TABLE 5 with values for morphine Sulfate and DAMGO included for comparison. Binding was determined in Chinese hamster ovary cell membranes as before. Antinociception (A50 values) was determined after i.c.v. or iv. administration using the mouse 55°C tail-flick assay.

Since the binding affinities and receptor pref- erences of the p-selective DAMGO derivatives are similar, the analgesic potencies of the glycopeptides are largely determined by their ability to penetrate the BBB by transcytosis [67], which in turn depends on the biousian character of the drugs [84]. One may consider two extremes that result in poor delivery of a peptide drug:.

■ The peptide binds tightly to biological membrane, effectively removing it from solution;

■ The peptide remains in aqueous solution, effectively preventing it from binding to biological membranes.

Thus, the goal in producing glycopeptides that are capable of effective CNS delivery, binding and GPCR activation, is to balance the degree of glycosylation, which effectively determines the amount of time the glycopeptide spends on the endothelial membrane of the BBB, as well as other membranes that the glycopeptide is likely to encounter. Affinity for the membrane is still required for effective binding and

activation of the GPCR but a certain amount of membrane hopping is required for effective drug transport. Thus, a plot of the BBB transport or antinociceptive A50 values versus the membrane affinity produced a U-shaped or V-shaped curve (FIGURE 6).

Unpublished studies in mice showed that disaccharide 29 produced behaviors (Straub tail and hyperlocomotion) suggestive of 'nar- cotic intoxication' at very low doses and had an extreme addiction liability as indicated by naloxone precipitated withdrawal studies. While p-agonists, such as peptide 26 or glycopeptide 27, could provide some useful clinical features that that morphine and other p-selective analgesics do not possess, there does not seem to be much appetite for adding drugs to the pharmacopeia with side-effect profiles worse than morphine, and that are likely to be extremely addictive.

Kaiser's pioneering studies on the

structure of ß-endorphin The late Emil Kaiser led a group of researchers at the Rockefeller Institute (USA) in studies of the structure and function of natutally occurring peptides, focusing their pioneering efforts on endogenous opioid hormones of mammalian origin including ß-endorphin [90] and other seemingly diverse peptides from arthropods such as bee venom, or melittin (FIGURE 7) [91]. In fact, both ß-endorphin and melittin interact strongly with biological membranes. It is in understanding the similarities and differences in exactly how these two peptides interact with

Table 7. Intrinsic helix stability in HO*.

Series Message sequence

Link

41 YtFGL 42 YtFGL 43 YtFGL 44 YtFGL 45 YtFGL 46 YtFGL 47 YtFGL

B = Aib = H 0

p

p

p

p

p

p

p

A = Ala =

Helical address sequence

NLBEKBLKS°'*'**L-NH2

NLBEKALKS0/*'**L-NH2

NLAEKBLKS°'*'**L-NH2

NLAEKALKS0/*'**L-NH2

NLAEKGLKS°'*'**L-NH2

NLGEKALKS°'*'**L-NH2

NLGEKGLKS°'*'**L-NH,

S° = OH

Wot soluble

12.2

7.4

7.2

8.7

7.1

8.1

H _H

Helicity per residue in H20 buffer (%)

S* = Glc

V H J V H

4.5

7.5

S** = Lact

0.7

1.0 7.0 1.3 6.9 0.1 0.0 0.0 0.8 0.0 0.7 0.0

'Variation in the intrinsic helicity was achieved with minimal impact on other properties by altering only two amino acid residues with B (Aib), A (Ala) or G (Gly). Water solubility could be affected by altering the glycosylation state of the S. Except for the extremely helical peptides without glycosylation, all of these compounds were highly water soluble, and showed only random coil behavior in aqueous solution. Aib: g-aminoidobutyric acid; P: Proline; S: Sen'ne.

' future science group www.future-science.com 219


Table 8. Amphipathic helix stability in the presence of micelles'.

Compound Message Link Helical address Helicity per residue in sodium sequence sequence dodecyl sulfate micelles (%)

S° = OH S* = Glc S** = Lact

41 YtFGL

42 YtFGL

43 YtFGL

P NLBEKBLKS°'*'**L-NH2 70 56.7 40.8

P NLBEKALKS°'*'**L-NH2

P NLAEKBLKS°'*/**L-NH2

60.8

58.5

44.9 35.7

35.7 33.6

44 YtFGL P NLAEKALKS°'*/**L-NH2 54.4 35.7

24.6

31.8

13.3 45 YtFGL P NLAEKGLKS°'*'**L-NH2 37.8

33.0 46 YtFGL P NLGEKALKS°'*'**L-NH2 14.5 4.5

47 YtFGL

B = Aib=^N'yNNl A H 0

P NLGEKGLKS°'*'**L-NH2 11.0 6.4 2.8

H 0 H 0

'Helix amphipath stability was greatly altered in the presence of sodium dodecyl sulfate by altering the two amino add residues with B (Alb), A (Ala) or 0 (Gly). Water solubility and the degree of helix formation (%-helix/residue) was also affected by altering the glycosylation state of the 5. Aib: a-aminoidobutyric acid; P: Proline; S: Serine.

membranes that will provide a rationale and opportunity for improving peptide drug delivery and effective design of CNS drugs. Our group has incorporated a design strategy for BBB penetration that exploits amphipathic a-helices that can interact strongly with cellular membranes to enhance endocytotic events. Critically, this biousian approach also preserves a degree of hydrophilicity for the overall peptide, especially when it is not interacting with the phospholipid membrane [84].

All of our glycosylated enkephalins display well-defined secondary structures in sodium dodecyl sulfate (SDS) micelles, irrespective of the identity of the glycoside attached to the peptide, which represent the simplest model for a biological membrane. In contrast, no

R = OLact 1 R = OGIc R = OH

Figure 11. Helicity in sodium dodecyl sulfate as a function of peptide address sequence and glycosylation state.

defined structure is observed in aqueous media (FIGURE 8A) [92]. Similarly, the longer glycopeptides related to ß-endorphin tend to adopt amphipathic helical structures in SDS micelles as well as phospholipid bicelles (FIGURE 8B) [93]. These larger glycopeptides displayed an ensemble of random coil conformations in aqueous solvent despite they are 17 residues in length. It is evident from these observations that amphipathicity of the glycopeptide (not simply hydrophilic or hydrophobic nature alone) is essential for membrane transport. This biousian behavior (i.e., random coil state in aqueous environment and highly folded state in membrane environment) appears essential for the effective transport of these larger peptides across the BBB [84].

Biousian behavior has also been observed for other endogenous GPCR peptide ligands [84.94]. Many, if not all peptide ligands that activate GPCRs lack a well-defined structure in aqueous buffer, but tend to fold into largely a-helical conformations in the presence of organic sol- vents [95] and lipid micelles [96], and in crystals [97,98]. Inooka and coworkers have been able to demonstrate that micelle-bound conformation of a peptide ligand is closely related to the actual receptor-bound conformation, that is, the a-helical region is similar in both cases [99].

Glycopeptide opioids based on endorphins Initially, we focused on the importance of the glycoside in the context of the short enkephalin-based glycopeptides to enhance membrane hopping and BBB penetration, as evidenced by

220 Future Mod. Chem. (2012) 4(2) future science group 1

Opioid glycopeptide analgesics derived from endogenous enkephalins & endorphins REVIEW

Table 9. Binding and intracerebroventricular antinociception as a function of helix stability - unglycosylated

i^^^nnm^ Compound Message

sequence Link Helical address

sequence 8 receptor

K, (nM)

Mreceptor K receptor

A50


41* YtFGL P

P

NLBEKBLKSL-NH2

NLBEKALKSL-NH2

16

9.6

8.7

2.3

27 1.66 42*/32

43*

44*

45*

YtFGL 2.8 0.57

YtFGL

YtFGL

YtFGL

P NLAEKBLKSL-NH2 9.7 10 28 0.18

P

P

P

P

NLAEKALKSL-NH2

NLAEKGLKSL-NH2

NLGEKALKSL-NH-,

NLGEKGLKSL-NH2

3.5 2.8 5.5 0.55

15

14

19

17

21

14

49 0.79 46*

47* YtFGL

YtFGL

23 0.96

17 1.92 IS m OH, |S* = G/c; 5 ** = Lact P: Proline; 5: Serine

peptide 35 compared with glycopeptide (TABLE 6) ;35]. The much larger endorphin-based peptides did nor always require glycosylation in order to achieve effective membrane hopping rates ro produce effective drug transport (FIGURE 9). In fact, the glycosylation could have no effect (32 vs 33), or even a deleterious effect on iv. potency (32 vs 34). We think that the flexibility of the linker (proline vs GABA vs DAVA) may play an important role as well, satisfying both the GPCR and the membrane binding requirements. More importantly although, the intrinsic stability of the membrane bound helix can decisively affect drug transport, as evidenced by iv. potency in vivo.

To determine the effect of helix stability a series of opioid C-terminal amide peptide 16-mers 41-47 were prepared using seven different helical segments with a minimal pertur- bation of the structure. Two amino acid residues (position 8 or 12) were substituted with one of three amino acids; helix-weakening glycine (Gly

or G); helix-preferring L-alanine (Ala or A); or strongly helix-preferring a-aminoisoburyric acid (Aib or B) to produce seven different peptides of decreasing helix stability (B-B, B-A, A-B, A-A, A-G, G-A and G~G). Each series of helices was prepared in three different glycosylation states; one peptide series with unglycosylated L-serine (S°); a series of monosaccharides bearing a single ß-O-D-glucose (Glc) on L-serine (S*); and a series of disaccharide glycopeptides bearing ß-0-lactose (Lact) on L-serine (S**). These closely related seven peptides and 14 glycopeptides were characterized by high field (600 MHz) NMR in the presence of SDS and D20/ H20, and by circular dichroism in H20, H20/ F3CCH2OH (data not shown here), and SDS/ H20 (FIGURE 10) [100,101]. The degree of helicity was conveniently expressed as%-helicity per residue [102,103]. Note that helicity was essentially absent in aqueous media (TABLE 7). Peptide 41 was not soluble in H20. However, in the presence of SDS micelles there was a clear trend,

Table 10. Binding and intracerebroventricular antinociception as a function of helix stability - glucopeptides.


Link Helical address sequence

K(nM) A5o


8 receptor u receptor K receptor

4I*

42*/33

43*

44*

YtFGL P NLBEKBLKS*L-NH2

NLBEKALKS*L-NH2

11 9.1 6.9 1.13 YtFGL P 3.6

14

7.9

6.3 5.7

7.1

2.3 2.6 0.58 YtFGL

YtFGL

P

P

P

P

P

NLAEKBLKS*L-NH2

NLAEKALKS*L-NH2

NLAEKGLKS*L-NH2

NLGEKALKS*L-NH2

NLGEKGLKS*L-NH2

6.1

6.2

2.2

12

8.9

6.3

1.46

1.44 45*

46* YtFGL

YtFGL 1.47

2.9

3.5

11 2.54 47* YtFGL

* = Lact 4.4 2.12

S = OH/IS* = G/c,|S P: Proline; 5: Serine



Table 11. Binding, intracerebroventricular and intravenous antinociception as a function of helix stability -


Link Helical address sequence 8

receptor

K.(nM)

M receptor

K

receptor

A5„ Intracerebro- ventricular (nmol)

A5„ Intravenous (pmol/kg)

4I** YtFGL P

P

P

P

P

NLBEKBLKS**L-NH2

NLBEKALKS**L-NH2

NLAEKBLKS**L-NH2

NLAEKALKS**L-NH2

NLAEKGLKS**L-NH2

5.9 1.2 2.5

5.7

3.5

0.57

0.11

0.21

4.3

1.1 42**/34 43**

44** 45**

46**

YtFGL

YtFGL

YtFGL

YtFGL

12

14

2.6

4.7

13

4.5

8.2 3.2

< 1

1.2

4.1 3.5 0.76

0.97 4.3 0.14

0.50

0.23

8.7

>40

YtFGL P NLGEKALKS**L-NH2 4.4

1.4

6.6

2.6 47**

S = OH; 5* = G/c P: Proline; 5: Sen

YtFGL P NLGEKGLKS**L-NH2

IS** = tacf.| ne.

(TABLE 8) with B-B strongly favoring helical conformations, G-G disfavoring helical conformations, and intermediate levels of helicity for the others in relatively smooth, although not monotonic, stepwise fashion (FIGURE II). The most helical compound, 41, was not soluble in water, but readily dissolved in the presence of SDS.

All 21 compounds were characterized in terms of 5/u/K-binding, and subjected to the 55°C tail-flick test in mice, both after i.c.v.

administration (TABLES 9-11). Administration with iv. bolus injection is currently being studied, and the data (not shown) is only preliminary. Although there were only modest variations in the binding selectivity, and in the A50 values obtained after i.c.v. administration (bypassing the BBB), there are large variations in the A50 values obtained after iv. injection, ranging from little or no antinociceptive activity observed at 32 mg/kg down to extremely potent activities with A_0 values below 500 ug/kg.

Executive summary

The three opiate receptors, p, 8 and K receptors, and their endogenous peptide neurotransmitters, the enkephalins and endorphins, play numerous roles in the CNS. Principal among these roles is the modulation of acute and chronic pain states. The endogenous peptide neurotransmitters are highly amphipathic, spending the bulk of their independent existence after cleavage from larger precursor proteins and prior to release stored in vesicles contained within the presynaptic membrane. Upon release they float in the postsynaptic membrane where they bind and activate opiate receptors. The effects of the endogenous peptide ligands are local in nature due to their poor transport properties.

Glycosylation of the relatively short enkephalins allows them to leave the membrane environment to engage in 'membrane hopping'. The key is to add a water soluble moiety to the peptide in such a way that in enhances water solubility without interfering with its interaction with the membrane. We have dubbed this effect 'biousian behavior' in which the glycopeptide may exist either in a relatively constrained membrane bound state, or an aqueous state as a 'random coil' ensemble. The degree of glycosylation can be adjusted to optimize the biological transport rates of the enkephalin-based glycopeptides to provide drugs based on the endogenous enkephalins.

The biousian behavior permits the glycopeptides and related serine phosphates to penetrate the blood-brain barrier, probably via transcytosis.

The larger endorphins have a message-linker-address peptide format. The message exists as a turn structure and is largely responsible for receptor binding and activation. The address exists as an amphipathic helix and is largely responsible for binding to the membrane. A flexible linker domain allows the receptor binding requirements and the membrane binding requirements to be met simultaneously. The address domain can be altered in order to optimize the biological transport rates of the endorphin-based glycopeptides based on the endogenous endorphins.

Peptide sequences can be varied to produce glycopeptide drugs that favor any of the three opiate receptors, or various combinations, such as U/K receptors and mixed u/8-agonists. Side-effect profiles of the analgesics can be manipulated by altering the agonist features of the enkephalins or the endorphin message segments.

Glycopeptide-based drugs have been developed that exert analgesic effects in mice far in excess of the classical narcotics, such as morphine, Demerol® and Oxycontin®. We are optimistic that interest in glycopeptide drugs derived from neuropeptides will increase, and that all major pharmaceutical companies will establish research programs in the area. We expect that the applications will not be limited to opiate agonists and that a large number of the 250 plus endogenous neuropeptides can be converted into glycopeptide drugs capable of penetrating the blood-brain barrier.



Future perspective While it would be premature to state that we have a complete understanding of the role of amphipathicity, biousian behavior [84] and membrane affinity in the transport of opioid peptides and glycopeptides, it is clear that the transport of these compounds across biological membranes of interest is not only possible, but quite efficient. It seems clear that the observed transport phenomena will not be limited to this class of neuropeptides, as researchers are exploring glycosylated endomorphins [104], dermorphins [105], dermorphins [106] and other peptides [107] with good results. Thus, we remain optimistic that interest in glycopeptide drugs derived from neuropeptides will increase and that all major pharmaceutical companies will establish research programs in the area.

Acknowledgements

We also thank D Guivalis, L St Louis, B Anglin and

L Szabb for assistance with the manuscript.

Financial & competing interests disclosure We thank the Office of Naval Research (N00014-05-01-

0807 and N00014-02-01-0471), the National Science

Foundation (CHE-6079I7) and the National Institutes of

Health (NINDS-NS-052727) for financial support. RPolt

and EJ Bilsky own founder's stock in Biousian Biosystems,

Inc., which holds intellectual property related to glycopep-

tide 25. The authors have no other relevant affiliations or

financial involvement with any organization or entity with

a financial interest in or financial conflict with the subject

matter or materials discussed in the manuscript apart from

those disclosed.

No writing assistance was utilized in the production of

this manuscript.

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